CAPTIVE BREEDING AND LARVAL REARING OF MAROON SPINY DAMSEL FISH, Premnas biaculeatus (BLOCH, 1790)

Transcription

1 Journal of the Andaman Science Association Vol. 19(1):78-87 (2014) ISSN , Printed in India Andaman Science Association, Port Blair (A & N Islands), India CAPTIVE BREEDING AND LARVAL REARING OF MAROON SPINY DAMSEL FISH, Premnas biaculeatus (BLOCH, 1790) S. Dam Roy, B. C. Ray*, Kamal Sarma, Grinson George Division of Fisheries Science, Central Island Agricultural Research Institute, ICAR, Port Blair A & N Islands, , India *Corresponding Author bipulray@yahoo.co.in ABSTRACT Captive breeding of marine ornamental fishes will be a boon for the marine ornamental fish industry. Therefore, a better understanding and rigorous approach to develop a breeding protocol is imperative for sustaining the industry and conservation of these fishes. Breeding of Premnas biaculeatus has been carried out in laboratory condition at Andaman for the first time. The egg tending activity was recorded visually by observing frequency of male and female reaching near the eggs for tending activity. The fertilized egg samples were collected, developmental stages were observed using microscope for embryogenesis study. 100% hatching was observed during the study and the larvae were separated from parents and stocked in larval tank which was connected to an air-lift bio-filter recirculatory system. Body colouration of P. biaculeatus started darkening from third day onwards and completed its larval stages after twentieth day. It was also estimated that about rotifers are required by a clownfish larvae per day for the better growth and survival. Keywords: Damsel fish, egg tending, embryogenesis, rotifer, spawning INTRODUCTION It is estimated that 1.5 to 2 million people worldwide keep marine aquaria (Green, 2003). Marine aquarium industry is of relatively low volume but of high value. Only about 10% of marine ornamental fishes are captivebred. Hence, till date wild collection is the main source of marine ornamental fish for international trade. However, wanton harvest of reef fishes is highly detrimental for the health of an ecosystem. Perciformes: Pomacentridae is an important group accounting for 43% of marine aquarium fish traded globally for years (Wabnitz et al., 2003). About 26 different pomacentrids are now being cultured worldwide. Premnas biaculeatus, maroon clown fish is one among this group gaining importance due to their aggressive behaviour and symbiotic association with species specific sea anemone. This species lives exclusively with Entacmaea quadricolor anemone. The density of anemone fish and their host anemone are good indicators of the state of a particular reef community as they are sparse in density and distribution (Sale et al., 1986) and therefore may be more prone to overexploitation (Couchman & Beumer, 1992). In Andaman Sea this species is readily available in shallow coral reef crest areas. There is no well organized ornamental fish trade prevailing in this island. But, considering its importance in the international ornamental fish trade as well as conservation, a better understanding on breeding behaviour of this species is imperative. The motivation for the present is to estimate the optimum environmental conditions for maturation, spawning and larval rearing of P. biaculeatus. The ontogeny of the embryo is also carefully observed to see the critical changes in the fish during its development in captive conditions. MATERIAL AND METHODS Brood stock collection P. biaculeatus lives in social groups in association with the anemone. In nature, among clown damselfishes, dominant and bigger fish is generally female and next 78

2 smaller fish is male (Madhu & Madhu, 2006). With this assumption, about 20 specimens of different sizes were collected from North Bay Reef of A & N Islands. Fishes were collected along with the associated anemone from a depth ranged from m during low tide hours using scoop net. Fish and anemones were packed separately in polythene bags, with a packing density of 2-3 fishes per bag containing 5 litres of sea water in aerated condition. In the laboratory collected fishes were maintained in separate quarantine tanks for a week. Later on healthy specimens were transferred to main hatchery system. Pair formation For pair formation, fishes were stocked together along with 3-5 sea anemones (Entacmaea quadricolor) in Fibre Reinforced Plastic (FRP) tanks of 500L capacity. Each tank contained 2-3 biological filters to maintain water quality. The biological filter was made of a circular conical plastic tub having 5-10 litres water holding capacity, where sand, pebbles and stones were arranged from top to bottom respectively. The thickness of the sand, pebbles and stones were 3 cm, 12 cm and 5 cm respectively. At the centre of the tub, ½ inch Poly Vinyl Chloride (PVC) pipe (40 cm length) was fitted with L shaped joint at one (upper) end and a small funnel at the other end embedded inside the filtering materials. A small hole was made in the PVC pipe and a tube with an air stone was fixed inside the funnel. This would give a flow of air-mixed water through PVC pipe from bottom to surface of the tank. Biological filter was dismantled, cleaned and dried once in every 15 days. Fishes were fed daily with shrimp, mussel and clam meat five times in a day. The feeds were thawed, chopped into smaller pieces and fed ad libitum. Fishes were also provided with rotifer Brachionus plicatilis and Artemia nauplii thrice in a week alternatively. Left over feeds were removed daily. Weekly 75 % water exchange was done. Fishes of desirable size and sex were allowed to form groups in order to form potential breeding pairs. After maintaining in this condition for a month, 5 compatible pairs of fishes were transferred to the brood stock rearing tanks. Brood stock rearing After pair formation, each pair was transferred and maintained separately in FRP brood stock tanks (100 l). A water depth of 40 cm was maintained in the tanks with plastic tub biological filter. One or two collected anemones (Entacmaea quadricolor) were also placed in the tank depending upon the size of the brooders. Filtered sea water was used every time for water exchange. Each tank was supplied with continuous aeration from a centralised air blower. Feeding was carried out similar as stated in pair formation. A porcelain tile was also placed at the bottom of the tank for attachment of eggs. Feeding for the parents after spawning and during incubation was restricted to a place so that eggs will not come in contact with the feeding materials. Embryogenesis study For embryogenesis study fertilized egg samples were collected using a dropper by mild scraping on the substratum from the spawning tank. Developmental stages were observed using microscope and photographed using a digital camera. The egg tending activity was recorded visually by observing frequency of male and female reaching near the eggs for tending activity and expressed as number of visits per minute. This information was recorded daily. Larval rearing After complete hatching, the tank was illuminated using a 60 watt bulb fixed above water surface at a corner of the tank. The height of the bulb from the water surface was adjusted in such a way that the temperature of water during larval rearing did not increase substantially. After hatching larvae were separated from parents, anemone and biological filter. The tank was connected to an airlift bio-filter recirculatory system. The system was made in an one tonne capacity round FRP tank like a simple biofilter. The only differences were it was of larger capacity and uplifting air-mixed water was allowed to fall into the larval rearing tank. An outflow drain pipe from larval tank was connected to the filter FRP tank which 79

3 automatically creates recirculation of water in between the tank systems. Bottom accumulation of the larval tank was removed daily as required by siphoning. Precaution was taken to prevent larvae escaping from the tank. Aeration was provided to meet the optimum dissolved oxygen concentration and air stones were placed in a hollow PVC pipe to avoid direct contact of air flow to larvae. Larval feeding Larvae were fed with rotifer and algae mixture as first feed. Locally available strains of rotifer Brachionus plicatilis was initially collected and cultured in mass scale by feeding exclusively on phytoplankton (Chlorella spp). Similarly mass culture of Chlorella spp was also continued on Conway media for feeding the rotifer. Feeding to larvae was done 4 times a day. Recirculatory system was temporarily stopped for an hour during feeding. However, to maintain oxygen level aerator line was operational. Water parameters Water quality parameters at the sites during brood fish collection, acclimatization tank, breeding tank and larval rearing tank were analyzed by following standard method (APHA, 1998). RESULTS AND DISCUSSION Gonadal growth, development, final maturation and spawning are the sequential processes in the reproductive development. If suitable husbandry and environment are provided, many fishes develop maturation process in captivity and spawn naturally (Olivotto et al., 2003). In the present study, utmost care was taken to provide suitable environmental conditions, feed and substrate to simulate natural conditions. Table 1: Water parameters recorded in collection site, different holding and rearing systems of P. biaculeatus Parameters Acclimatisation tank Breeding tank Larval tank Brooder collection site Temperature ( 0 C) Salinity (ppt) ph Dissolved oxygen (ppm) Alkalinity (ppm) Out of 5 pairs of brood fishes collected, acclimatized, paired and finally reared in conditions (Table 1), one pair (Fig. 1) spawned voluntarily after five months in the laboratory condition. The Average weight and length of females and males were 31.13±6.92 gm; 10.16±0.64 cm and 6.92±1.64 gm; 6.56±0.59 cm respectively. Previous studies reported that the damsel fishes generally spawn within months in captivity after pair formation (Dhaneesh et al., 2009, Olivotto et al., 2003), however, some fishes took 4-6 months to start spawning (Madhu et al., 2006). The weight and length of the female of breeding pair was gm and 11.4 cm and male was gm and 8.0 cm respectively. It was interesting to note that this breeding pair was the biggest in size compared to the other four pairs. Few days before the onset of the spawning process, breeding pair moves together closely and horizontally, frequently cleaning and fanning a specific site (substratum) of the tank for releasing the eggs. Spawning was observed in the early morning hours which lasted for 30 to 60 min. It is observed that in most occasion spawning occurred after water exchange, within h of water exchange. During egg laying process, female touches her genital organ on the selected site and released eggs in batches. These eggs were simultaneously 80

4 fertilised by the male. Generally female released the eggs in circular cluster form just under the margin of the stinging tentacles of the host sea anemone. This might be a way to safeguard the eggs from potential predators. In nature also P. biaculeatus lay benthic eggs, males are generally responsible for nest site selection and nest tending (Clutton-Brock, 1991). The fertilized eggs were separative in nature, oval shaped, capsule like and bright orange in colour (Fig. 1). These eggs got attached to substratum with the help of mass of hair like adhesive threads, perpendicularly hanging in the water column till they hatched out. In general most of the benthic spawners attach their eggs to the substratum, often in discrete monolayer clutches (Robertson, 1991). Fecundity of the first spawning of the breeding pair was less (210 numbers), however, in the subsequent spawning the number of eggs increased substantially (average of last 6 spawning = 2148 ± 252 nos). Till date, spawning was observed 7 times from June to October 09 and spawning occurred approximately within an interval of three weeks ( days) with a range of days. Developmental stages or embryogenesis (Table 2) of eggs lasted for 6½ days and during that period parental care was observed. Though during incubation there was no significant difference in egg tending activity (Fig. 24) between sexes (p < 0.05), activity was fluctuated as embryonic development progressed (p > 0.05). The highest activity was observed in the first day and possibly due to exhaustion the activity was lowest on the second day. From 5 th day onwards activity progressively increased. For the egg tending purpose, both the parents took active part in fanning, cleaning and mouthing of eggs (Fig. 1). However, egg tending activity by the male superseded the female from the 5 th day onwards and on the final day the activity was considerably increased (p < 0.05). Unlike the present study, male generally spend more time than female in active egg tending (Allen, 1980; Wilkerson, 1998). All these activities are very much necessary for survival and development of the eggs, like fanning by the pectoral and caudal fin increases the oxygen availability to the eggs. Green (2003) opined that main objective of fanning is to increase oxygen to the eggs as he found low level of oxygen when parents were removed from the tank. Fanning is also important for removal of debris, disturbing the boundary layers of the eggs (St. Mary et al., 2001) as well as to remove metabolic wastes from the cluster (Keenleyside, 1991). Mouthing behaviour is also important to remove dead larvae and cleanse live ones (Keenleyside, 1991) with an antimicrobial compound within the parental epidermal mucus (Knouft et al., 2003). Dissolved oxygen (DO) and temperature are of prime importance in fish metabolism during the early development of fishes (Rombough, 1988; Jobling, 1995). In the brooder tank, temperature within a range of o C and DO within 6-7 mg/l were prevailing throughout the growing period (Table 1). The other parameters like salinity (30 ± 1 ppt), ph ( ) and alkalinity ( mg/l) were similar to the condition of the sea from where brood fishes were collected. Microscopic observations revealed that egg yolk was three fourth of the size of the eggs, having numerous oil globules in it (Fig. 2). Average length of eggs (longest side) was ± 0.02 mm. The egg size was less in comparison to other damsel fishes ( mm). In case of Amphiprion chrysogaster much bigger size (2.9 mm) was observed, however, the incubation period was 8 days and fecundity (400) (Durville et al., 2004) was much lower than the present study. Thus it can be assumed that species variation, high fecundity and long incubation period, repeated spawning of the same pair might be some of the possible reasons for smaller size of the eggs. After fertilization, embryogenesis proceeds with the transformation of the embryos from yolk dominated larvae with significant morphological and functional development (Green, 2004). Hatching process started 152 hr after fertilization, mostly during evening hours and all the eggs hatched out within 159 hr (Fig. 21). During the study 100% hatching was observed, as no eggs were seen attached to the wall after hatching process is completed. However, actual hatching percentage could not be ascertained owing to parental cleaning and fanning by which bad and damaged eggs were removed from the attachment. 81

5 82

6 Table 2. Embryonic development stages of P. biaculeatus Days Figures Developmental stage 1 st day Fig. 1 Newly spawned eggs with tending parents. Fig. 2 The eggs were spherical in shape, contain numerous oil globules within the large yolk mass. The egg yolk mass and the animal pole area were distinctively visible and separate. Fig. 3 First cell division was observed min after fertilization. Cell division started from the stalked end of the eggs. The separating process initiated in the cytoplasm of blastodisc in to two blastomeres. Fig. 4 Second cell division ( 4 cells) took place within min after fertilization. Fig cell division was observed after 3 h 16 min. Fig. 6 Blastula formation took place in 7 ½ h. In this condition the blastomeres were very small and look like a mass of cells. Fig. 7 Gastrulation process started and two separate layers were formed above the yolk mass. The cells or blastomeres were slowly moved towards the vegetal pole engulfing the yolk mass (11 12 hr ) Fig. 8 Gastrulation completed within 24h and embryogenesis began. Rudimentary tail/ trunk bud formation started appearing from the animal pole (anterior most point of the gastrula). Number of oil globules decreased. 2 nd day Fig. 9 Within h melanophore also started appearing on the yolk mass. Head demarcation and tail clearly visible, engulfing the ¾ th of yolk mass. Immediately after head a notch was formed and spinal cord started developing. Tail somite division was also clearly visible. The head was slowly moving towards the opposite direction. The egg colour changed from orange to brown. Fig. 10 Within 48 h vertebrate column becoming clearly distinctive within the somatic cell formations. Anterior most end of spinal chord has been enlarged to form the head. Both the sides of the head with colourless eye socket were formed. Pericardial area was clearly visible. Tail portion became free from the yolk mass. 3 th day Fig. 11 On the third day onwards the rudimentary heart valve was clearly distinct and beating, creating a wave like motion. In the latter half of the day two chambered heart started circulating blood along the circumference of yolk mass and in the spinal cord, leading to brain and back to the heart. Eyes darkened and appears as heart shape. Fig. 12 The segmented head was distinctly visible. Blood circulation was more prominent and blood cells were moving like in a mass. Rudimentary pectoral fin was visible. 83

7 4 th day Fig. 13 ( 84 h) Fig. 14 (88-90 h) Fig. 15 (96 h) The embryo getting enlarged occupying whole eggs of capsule. Head segmentation was in process. Rudimentary gill arch like structure was visible behind the eye ball. Blood circulating through that area. Only few large oil globules can be visible in the yolk mass and size of the yolk mass was decreasing. Pectoral fin was quite large and distinct, flickering occasionally. Optic nerve was visible. Head segmentation almost completed. In the later part of the day three gill arch like structures could be seen and blood movement through that area. Close blood circulation was observed throughout the embryo. Rudimentary gut cavity started appearing bordering the yolk sac in the latter half of the fourth day. 5 th day Fig. 16 & 17 (120 h) Gut cavity was more prominent. Body twisting movement was more. Three rudimentary gill arches were clearly visible. The yolk sac reduced near the gut area. Body segmentation was distinctly visible and also some fin rays. In the later part of the development (Fig. 16) transparency decreased. 6 th day Fig. 18 (144 h) Gut cavity was very clear. Eye became circular and distinctly black. 4 gill arches were visible and blood flowing through this area. The transparency of the eggs further decreased with the development of the eggs. Organs became enlarged. Head almost becoming one third of the egg capsule. 7 th day Fig. 19 & 20 Eggs were becoming more or less opaque. Few anal fin rays can be seen. Aorta was distinctly visible. Twisting movement increased as time passed. Hatching started from 4.30 pm (152 h) onwards and by 7.0 pm ( 154 h) about 50% hatched out and by 12 pm (159 h) all hatched out. Before hatching embryo showed vigorous movement and tail portion first got detached and slowly body comes out of the capsule. It takes about 12 h to complete hatching of the eggs. 8 th day Fig. 21 Immediately after hatching larvae came out with the yolk sac and oil globules. Few melanophore on the body along the line near the vertebrae and near the gut area. The dorsal, caudal and anal fins were continuous longitudinally. The size of the larvae was 3.72 mm. These larvae were free swimming in the water column, however the movement was slow. 9 th day Fig. 22 After yolk absorption. Melanophore intensity increased both on the line along the vertebrae and near the gut area. >12 day Fig. 23 Completely metamorphosed larvae with distinct two vertical white colour bands. 84

8 Fig. 24. Egg tending activity of P. biaculeatus during incubation period. Newly hatched larvae were having an average total length of ± 0.05 mm and were relatively active. They were transparent in nature with distinct visible black eyes and a small yellowish-black yolk-sac. A continuous light source (60 watt bulb for 20 days) was provided with an assumption that fish could grow faster due to longer feeding period owing to extended photoperiod of 24 h (Olivotto et al., 2003). Initially they moved slowly in the water column as they are heavy due to yolk sac. However, the yolk-sac was absorbed within 24 h (Fig. 22) and they actively swam near the surface of the water. After yolk absorption, larvae were more active swimmers and visual feeders, mostly feeds on rotifer. Later on the larvae were fed with newly hatched Artemia nauplii and wet feeds (Table 3). Body colouration of P. biaculeatus started darkening from 3 rd day after hatching. From fifth day onwards, light orange colour started appearing in the body of the larvae. On the next day (6 th day) light orange colouration almost became permanent and appeared exactly like the original colour of the species. On the 12 th day after hatching, two vertical white colour bands started appearing on the dorsal part of the body, one just posterior to head and another in the caudal peduncle. From th day onwards third band started appearing in between the earlier two bands. Slowly these bands broadened with the growth of the fish. In the present study survival percentage of the larvae was very low (< 6%). The poor survival of larvae may be due to inadequate supply of rotifer as well as poor nutritive value of the rotifer supplied. It is estimated that about rotifers are required to be consumed by a clownfish larvae per day for the better growth and survival (Wilkerson, 1998). Olivotto et al., (2003) reported that fish larvae do not survive if fed on rotifers only, which in turn, were fed only on algae. They opined that proper rotifer nutrition and enrichment represents the underlying foundation for the successful rearing of larvae. Starving or poorly nourished rotifers are inadequate as first food. In the present study continuous illumination might be another reason to impart stress on fish larvae and resulting in poor survival of the larvae. 85

9 Table 3: Feeding schedule for larval rearing Days after hatching Feed type Feeding schedules 1 st day/after yolk-sac absorption Algae + rotifer mixture 50,000 nos/ml nos/ml 2 nd - 14 th day nos/ml 15 th -30 th day Rotifer+ newly hatched Artemia nauplii 30 th day onwards Rotifer + newly hatched Artemia nauplii +fish egg granules 5-7 nos/ml, Artemia 3-5 nos/ml 5-7 nos/ml, Artemia 3-5 nos/ml, fish egg granules fed till satiation. Coral reef ecosystem is very delicate and fragile. In the recent years there is a surge in the trade of ornamental fishes. Indiscriminate harvesting of ornamental fishes not only endangers the survival of these species but also the reef ecosystem as a whole as ornamental fishes are important partners of the self sustained reef ecosystem. Therefore, a better understanding and rigorous approach to develop a breeding protocol is imperative not only for sustaining the industry but also as a matter of conservation of these fishes. The results of present study will be also useful for general maintenance of the brood stock, their spawning and embryogenesis. However, optimization of larval rearing protocol is of primary importance for development of the commercial production of this elegant species. REFERENCE Allen, G.R. (1980). The Amenonefishes of the world. Species, care and breeding. Vol. Aquarium Systems, Ohio APHA-AWWA-WEF. (1998). Standard Methods for the Examination of Water and Wastewater. In: Clesceri L S, Greenberg A E, Eaton A D (ed) American Public Health Association, American Water Works Association, Water Environment Federation, Washington DC. Clutton-Brock, T.H. (1991). The evolution of parental care, Vol. Princeton University Press, NJ. pp In: Complex Ecosystem, (Eds.) Sale, P., Academic Press, San Diego CA, USA Couchman, D. & Beumer, J.P. (1992). The commercial fishery for the collection of marine aquarium fishes in Queensland: status and management plan. Department of Primary Industries, Queensland. Information Series Q Dhaneesh, K.V., Ajith Kumar, T. & Shunmugaraj, T. (2009). Embryonic development of clown fish Amphiprion percula (Laceped 1802). Middle-east journal of scientific research, 4: Durville, P., Fabre, J.N. & Germain, G. (2004). The breeding of clown fish Amphiprion chrysogaster (Cuvier, 1830) endemic to the Mascarone Islands (Indian Ocean). Aquarium systems manufacturer of instant ocean. pp Green, B.S. (2004). Parental and environmental effects on the early life history of a tropical fish Amphiprion malanopus. Ph.D dissertation, School of Marine Biology and Aquaculture, James Cook University, Queensland, Australia. Green, E. (2003). International trade in marine aquarium species: using the Global Marine Aquarium Database. pp In: Marine Ornamental Species: Collection, Culture, and Conservation, (Eds.) Cato, J. & Brown, C., Iowa State Press, Ames, USA. Jobling, M. (1995). Environmental Biology of fishes. Chapman and Hall, New York. Keenleyside, M. (1991). Parental care. pp In: Cichlid fishes: behaviour, ecology and evolution, (Eds.) Keenleyside, M., Chapman and Hall, London. 86

10 Knouft, J.H., Page, L.M. & Plewa, M.J. (2003). Antimicrobial egg cleaning by the fringed darter (Perciformes: Percidae: Etheostoma crossopterum): implications of a novel component of parental care in fishes. Proc Biol Sci., 270: Madhu, K. & Madhu, R. (2006). Protandrous hermaphroiditism in the clown fish Amphiprion percula from Andaman and Nicobar Island. Indian J Fish., 53(1): Madhu, K., Madhu, R., Krishnan, L., Sasidharan, C.S. & Venugopalan, K.M. (2006). Spawning and larval rearing of Amphiprion ocellaris under captive condition. Mar Fish Infor Serv, T & E Ser, No 188pp Olivotto, I., Cardinali, M., Barbaresi, L., Maradonna, F. & Carnevali, O. (2003). Coral reef breeding: the secretes of each species. Aquaculture, 224: Robertson, D.R. (1991). The role of adult biology in the timing of spawning of tropical reef fishes. pp In: The ecology of coral reef fishes, (Eds.) Sale, P., Academic Press, New York. Rombough, P.J. (1988). Respiratory gas exchange, aerobic metabolism, and effects of hypoxia during early life. pp In: Fish Physiology. The Physiology of developing fish, eggs and larvae, Vol XI, (Eds.) Randall, W.S. & Hoar, D.J., Academic Press, New York. Sale, P.F., Eckert, G.J., Ferell, D.J., Fowler, A.J., Jones, T.A., Mapstone, B.D. & Stell, W.J. (1986). Aspects of the demography of seven species of coral reef fishes, with recommendations for their management. Report to the Great Barrier Reef Marine Park Authority, Townsville, Queensland. St. Mary, C., Noureddine, C. & Lindstroem, K. (2001). Environmental effect on male reproductive success and parental care in the Florida Flagfish, Jordanella floridae. Ethology,107: Wabnitz, C., Taylor, M., Green, E. & Razak, T. (2003). From Ocean to Aquarium. UNEP-WCMC, Cambridge, UK. 60 p. Wilkerson, J.D. (1998). Clownfishes. A guide to their captive care, breeding and natural history, 1st ed, Microcosm, Shelburne, VT. 240 p. Publish With Us 87